Lyme disease is the most common vector born infectious disease in North America, Asia and Europe. It is a multisystem, inflammatory, progressive disease with a wide range of clinical manifestations, sometimes including erythema migrans (“EM”) the initial and readily recognized cutaneous “target” lesion. From EM, which may only present in an estimated 23% of the patients, it may disseminate to other organs, including the nervous system, joints, and heart. Such progression may result in permanent neurological and/or musculo skeletal damage, and debilitating symptoms including fatigue and other flu-like symptoms.
A diagnosis of LD is currently based on clinical symptoms and serology, i.e., antibody to LD. The latter may not be detectable during the first few weeks of infection. The current LD diagnosis methodology uses a recommended two-tier serological assay, misses up to 60% of early infections, and is unable to distinguish a past infection from current, active Borrelia infection. More specifically, current sero-diagnostic assays include an ELISA assay to detect antibodies to Borrelia species followed by a Western blot for confirmation. If diagnosed in the early stages, the disease can generally be cured with therapeutic agents, e.g., antibiotics. If left untreated, complications involving joints, the heart, and the nervous system can occur. It is therefore crucial to be able to specifically detect and diagnose Lyme disease at an early stage in order to avoid complications that may develop in later stages. The instant invention includes a method that employs two or more biomarkers to diagnose and treat early LD, where early LD is defined at less than or equal to eight weeks from infection/exposure.
Due to the fact that few spirochetes are present, i.e., Borrelia burgdorferi, the spirochete that causes LD, especially in blood specimens, the best current methods measure immune response. While not fully reviewed here, many steps, including antigen capture by “professional antigen presenting cells” and numerous signaling and processing steps, as well as coordination of various immune cell types, are required prior to production of Ab. For example, prior to Ab secretion numerous immune signaling agents must be secreted and transported from one type of immune cell to another. An immune network including APCs, T-cells of various types and B-cells is generated. Even once these signals are generated numerous steps of B-cell maturation, transcription, translation, processing and secretion are required before even low levels of IgM Ab are present in the blood. Evolution has driven the spirochete to attempt to evade and suppress the Ab response, and numerous steps between the APC and the Ab provide many possible opportunities for suppression. Certain subjects, i.e., genotypes, may also be less effective in promptly completing the process and producing high levels of Ab. Thus, measurement of the earliest steps of immune response, prior to antibody excess that can be measured in the blood, is rationally expected to a more sensitive method of detecting early infection.
Although the Ab response may be delayed or weak, a significant number of acute Lyme disease patients have such a florid early immune response that it may be visible with the naked eye as erythema migrans (EM). It is known that some clinicians may miss EM, and some skin types may not show the response. Further, it should be noted that EM detected clinically may not be indicative of Lyme disease. For examples of descriptions and differential diagnosis based on apparent EM, see, e.g., Hsu (2001) Am Fam Physician 64(2):289; and, Nopper (1998) Pediatr Ann 27:136. Other symptoms of initial Lyme disease including, fever, malaise, arthralgia, headache and stiff neck are even less specific than EM.
The significant disadvantages (poor sensitivity and specificity) of current assays lead to a significant medical need for better diagnostic tests for diagnosing and treating early Lyme disease. There are some methods that culture blood cells and look for cytokine production in response to antigens for the detection of LD, however these are expensive, labor intensive, rely on artful methods and require living white blood cells. Molecular methods of identifying the immune mediators that can “see” the early immune response, even when the naked eye, historically an insensitive system, cannot see a skin lesion, are targeted in the present invention because molecular methods typically are more sensitive than clinical observation in detecting immune responses.
Hence, there remains a need for early identification and/or diagnosis and/or treatment of LD via molecular methods, and specifically via biomarkers that can be easily assayed using ELISA type assays common in the clinical lab. Identification of biomarkers associated with LD may also aid in identification of key molecular pathways that may be targeted for therapeutic purposes. More specifically, biomarker analysis can provide prognostic as well as diagnostic information, guide initial treatment choice, monitor treatment efficacy, and improve outcomes. The two or more biomarkers, hereinafter referred to as the biomarker signature, of the instant invention allow specifically for early disease diagnosis, effective treatment, and progression prevention. Early diagnosis, appropriate treatment, and prevention of progression and/or recurrence, and biomarker testing can play a role in all of these management areas. The poor sensitivity and specificity of the current methods for detecting acute LD means that methods for the diagnosing, prognosing, monitoring, differentiating, treating, and managing of Lyme disease in a subject characterized by the detection of a biomarker signature comprised of a combination of two or more analytes indicative of disease would be an invaluable tool to aid clinicians. Such methods have the potential to expedite and increase the accuracy of LD diagnosis and treatment.
A method such as discussed herein, that uses, for example, serum, plasma, blood, blood spots, blood filtrate, urine, saliva or tears, detects the in vivo production of the biomarker signature, i.e., cytokine or other analyte markers, would be easier, more generally applicable, and more accurate that those currently available. Both highly specific and more sensitive tests detecting biomarker signatures may also have value as a component of a multi-tier LD assay. Such a biomarker assay would be more sensitive and more specific than current serological assays, and would provide detection and diagnosis at early time points, i.e., earlier than detection of antibodies. The present invention is an effective diagnostic method for LD that improves disease outcomes in patients through early detection and supporting early treatment.